Plasma heavy metal levels correlate with deregulated gene expression of detoxifying enzymes in osteoporotic patients

Heavy metal levels appear to be associated with low bone mineral density (BMD) and the consequent osteoporosis risk, but the relationship with the disease has not been clearly defined. The altered expression pattern of numerous genes, including detoxifying genes, seems to play a pivotal role in this context, leading to increased susceptibility to several diseases, including osteoporosis. The purpose of this study is to analyse circulating heavy metals levels and the expression of detoxifying genes in osteoporotic patients (OPs, n = 31), compared with healthy subjects (CTRs, n = 32). Heavy metals concentration in plasma samples was determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and the subsequent expression analysis of NAD(P)H quinone dehydrogenase 1 (NQO1), Catalase (CAT), and Metallothionein 1E (MT1E) genes in Peripheral Blood Mononuclear Cells (PBMCs) was assessed by real-time polymerase chain reaction (qRT-PCR). Copper (Cu), mercury (Hg), molybdenum (Mo) and lead (Pb) were found to be significantly higher in the plasma of OPs compared to CTRs. Analysis of the expression levels of detoxifying genes showed a significant decrease in CAT and MT1E in OP group. In addition, Cu correlated positively with the expression levels of both CAT and MT1E in CTRs group and MT1E in OPs. This study shows an increased circulating concentration of certain metals combined with an altered expression pattern of detoxifying genes in OPs, highlighting a novel aspect to be investigated in order to better characterize the role of metals in the pathogenesis of osteoporosis.

Heavy metals such as Lead (Pb), Mercury (Hg), Arsenic (As), and Cadmium (Cd) have been classified by the World Health Organization (WHO) as global environmental pollutants that require constant monitoring given their numerous adverse health effects 1,2 . In fact, exposure to these pollutants can affect cardiovascular, endocrine, respiratory, reproductive, and immune systems 3 . Heavy metals are also considered capable of impairing the functionality of several proteins, and modulating the expression pattern of numerous genes, altering physiological processes and thus leading to increased susceptibility to the development of certain diseases 3 . The chemical properties of heavy metals allow them to interact with the cell's macromolecules, leading to their oxidative deterioration 4 . According to literature data, long-term exposure to heavy metals and their bioaccumulation can induce oxidative stress and also lead to dysregulation of detoxifying genes expression levels, which play a primary role in the elimination of Reactive Oxygen Species (ROS) 5 . It has been reported that Pb and Cd induce lipid peroxidation, impairing the functionality of key cellular anti-oxidant mechanisms by decreasing the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels 6,7 . According to further studies, in addition to Pb and Cd, As, Hg and Nickel (Ni) are also responsible for redox reactions that occur in major biological systems, leading to the production of free radicals that damage proteins and DNA 8 . As already mentioned, heavy metals can accumulate in different body districts, including bone tissue. Indeed, there are several studies reporting a correlation between Pb and Cd blood levels and Bone Mineral Density (BMD), with consequent increased risk of fragility fractures 9 . Absorbed Pb circulates in the plasma and then is exchanged www.nature.com/scientificreports/ mainly between different tissues 10 . Bone is capable of storing Pb, which can be subsequently mobilized and can generate neurotoxic and teratogenic effects, respectively 11 . Environmental exposure to Cd can also affect bone homeostasis, leading to osteomalacia, osteoporosis and Itai-Itai diseases 12 . Therefore, it has been shown that Pb and Cd can exert direct effects on bone cells by affecting the processes of bone tissue apposition and resorption. To date, Al-Ghafari and colleagues report how treatment of osteoblast cultures with Pb and Cd reduces cell viability, impairing bioenergetic processes resulting in reduced levels of ATP produced 13 . The hypothesis of a potential involvement of heavy metals in bone tissue remodelling is also supported by the reports of Scimeca and colleagues, who observed accumulation of Pb, Cd, and Chromium (Cr) in femoral head biopsies of osteoporotic patients. The presence of these metals appeared to correlate with Sclerostin levels detected in the same tissues, suggesting the presence of a molecular link between heavy metal bioaccumulation and dysregulation of bone metabolism in osteoporosis 14 . Therefore, the aim of the present study was to analyze plasma levels of major heavy metals in osteoporotic patients (OPs) and healthy subjects (CTRs). In addition, circulating levels of heavy metals were correlated with the expression levels of genes encoding for three enzymes involved in the body's detoxification processes, including NAD(P)H quinone oxidoreductase (NQO1), Metallothionein 1E (MT1E) and Catalase (CAT). Data obtained highlight the pivotal role played by metals in osteoporosis, in association with the altered expression pattern of enzymes mainly involved in the defence against oxidative stress, which is essential for maintaining the homeostasis of the musculoskeletal system.

Materials and methods
Subjects. The study was approved by the Ethical Board of the Azienda Ospedaliera Universitaria "Policlinico Tor Vergata" (approval reference number #17/21). Subjects included in the study were divided into two groups: 31 OPs undergoing surgery for low-energy fragility fracture and 32 CTRs undergoing surgery for high-energy fracture. Subjects with malignancies, endocrine disorders affecting bone and mineral metabolism, autoimmune diseases and bone disorders other than primary osteoporosis, as well as those on long-term therapy with drugs that interfere with bone metabolism, sex hormone replacement therapy and antifracture and/or osteoanabolic therapies, alcohol abuse and HBV, HCV or HIV infections were excluded from the study. A questionnaire was administered to all subjects recruited in the study to collect information of their habit of smoking. All participants signed informed consent before surgery, and all experimental procedures were performed according to the Code of Ethics of World Medicine (Declaration of Helsinki).
Clinical and biochemical parameters. Dual-energy x-ray absorptiometry (DXA) was performed with a Lunar DXA apparatus (GE Healthcare, Madison, WI, USA). Scans of the lumbar spine (L1-L4) and femur (neck and total) were performed, and BMD measurements (g/cm 2 ) were recorded along with t-scores, with a coefficient of variation of 0.7%, according to the manufacturer's recommendations. Finally, Calcium (Ca), PTH and 25-(OH)-VitD levels were measured in fasting venous blood samples 15 . Detailed clinical characteristics of the study subjects are summarized in Table 1.

Specimen collection.
Peripheral blood (4 ml) was collected in EDTA tubes the day after surgery. Blood was placed in a falcon containing Ficoll-Paque™ PLUS and centrifuged at 400×g for 20 min at 4 °C in a brake rotor. Subsequently, the layer appearing at the interface between plasma and sediment after centrifugation was carefully removed and inserted into a new tube. Finally, two washes were performed with 1X Phosphate-Buffered Saline (PBS) by centrifugation at 200×g for 10 min at 4 °C. Peripheral Blood Mononuclear Cells (PBMCs) pellet was resuspended adding 1 ml of TRIzol reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and immediately frozen at 80 °C until further processing. Statistical analyses. Data were analyzed with GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, USA). Before applied statistical procedures, the assumptions of normality were checked for each variable using the D' Agostino-Pearson test. A non-parametric Mann-Whitney U-test was used for variables showing a skewed distribution, whereas data following a normal (Gaussian) distribution were processed with an unpaired Student's t-test. The correlation analyses between clinical/biochemical data, qRT-PCR and iCAP-RQ were evaluated using the Spearman's rank test. Data are presented as the mean ± standard error mean (SEM). Differences were considered significant when the p value was < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 **** 15 .
Ethics approval. The study was approved by the Ethical Board of the Azienda Ospedaliera Universitaria "Policlinico Tor Vergata" (approval reference number #17/21). All experimental procedures were performed according to the Code of Ethics of World Medicine (Declaration of Helsinki).

Consent to participate.
Informed consent was obtained from all individual participants included in the study.
Expression profile analysis of detoxifying NQO1, MT1E and CAT encoding genes. In order to investigate whether heavy metal exposure altered the expression levels of detoxifying genes, the mRNA expression levels of NQO1, MT1E and CAT in OPs group and CTRs were quantified by qRT-PCR. No significant difference was found in the NQO1 expression levels between OPs and CTRs (1.18 ± 0.13 vs 1.30 ± 0.25, p = 0.3698). Conversely, we found a significant inhibition of MT1E and CAT mRNA expression levels in OPs compared to CTRs (0.88 ± 0.07 vs 1.18 ± 0.12, p = 0.0349 and 0.78 ± 0.08 vs 1.36 ± 0.18, p = 0.0059, respectively) (Fig. 2).

Correlation between plasma concentration of heavy metals and PBMCs detoxifying genes expression levels.
To identify possible correlations between circulating levels of heavy metals and altered  www.nature.com/scientificreports/ expression pattern of detoxifying enzymes in OPs, a correlation analysis was conducted. By applying the Spearman correlation test, a statistically significant direct correlation was identified between Cu levels and the expression levels of both CAT and MTE1 in CTRs (p = 0.0499 and p = 0.0212, respectively). In addition, a statistically significant direct correlation was identified between Cu levels and MTE1 expression in OPs (p = 0.0318) (Fig. 3).

Discussion
Trace metals are ubiquitous environmental pollutants with toxic properties 17 . Human exposure to these chemicals may occur occupationally, environmentally, or through dietary intake. In the general population, the main risk factors are represented by food and water, while cigarette smoking is an additional relevant source of exposure to trace metals 18 . Contaminants in food and water enter the body through absorption by the gastrointestinal tract, as is the case of Pb, Cd, and As. Pb and As can also enter through the skin, while Cd enters the respiratory tract through inhalation 18 . The exposure of metals is capable of causing deleterious effects on all systems of the human body. Several in vivo and in vitro studies show how heavy metals can affect bone homeostasis, inhibiting skeletal growth and deposition of new bone matrix, thus leading to degenerative diseases, including osteoporosis and osteoarthritis 19 . The present work aims to identify the role of circulating metal levels in OPs, investigating their essential involvement in the pathogenesis of the disease and highlighting the need to further study their role in osteoporosis. There are growing literature data that heavy metals result in altered homeostasis of bone tissue, leading to the development of chronic degenerative diseases such as osteoporosis and osteoarthritis 9,20 . A recent study analyzed the association between the presence of toxic metals in serum and BMD in 60 male adults, showing a correlation between blood Pb and Cd levels and the risk of osteoporosis 21 . Interestingly, blood levels of both metals correlated negatively with blood levels of Ca, suggesting a potential involvement of Pb and Cd in modulating BMD and consequently representing an important risk factor for the onset of osteoporosis. Confirming the potential role played by metals in bone metabolism, Sadeghi et al. identified increased bioaccumulation of Zn, Cu, Pb and Cd in the plasma of 49 osteoporotic women. Although the result was not statistically significant, the differences were greater in patients with more severe disease (t-score < − 1.7) and the presence of the metals correlated negatively with the femur t-score 22 . Confirming these preliminary data, our results broaden the landscape of the role played by metals in osteoporosis by identifying higher concentrations of Cu, Mo, Hg, and Pb in the plasma of OPs compared to CTRs. It is known that excess Cu can influence generalized BMD loss, causing rickets and abnormal osteophyte formation in patients with Wilson's disease 23 . Furthermore, as reported by Qu and colleagues, elevated serum Cu levels were found to be significantly associated with increased osteoporosis  www.nature.com/scientificreports/ fractures, especially in men, supporting our results 24 . Pb, on the other hand, represents an important environmental pollutant, and several studies have shown that acts negatively on osteoblasts and osteoclasts, delaying the healing time of fractures by inhibiting the progression of endochondral ossification 25 . In addition, it was often found that bone tissue from OPs with increased Pb presence had lower histomorphometric Bone Volume (BV/ TV) values 14 . Finally, the effect exerted by bioaccumulation of Hg and Mo on bone has not yet been elucidated 23 . Although bioaccumulation of metals is now known to exert negative effects, either directly or indirectly, on bone tissue, studies investigating the potential role played by bioaccumulation of Hg on BMD are conflicting and in most do not report statistically significant correlations 26 . Wei et al. investigated the effect of some dietary intake metals on BMD, reporting that co-exposure to Pb, Cd, Hg, Selenium (Se), Manganese (Mn), and Zinc (Zn) correlated with decreased BMD, however, only Pb, Mn, and Se were identified as the main contributors to this association 27 . Our results also report elevated serum levels of Mo in OPs, compared to CTRs. In agreement with this finding, Lewis et al. 28 identify an inverse association between the amount of Mo in urine and BMD of both the lumbar spine and femoral neck, suggesting that this metal may impair bone metabolism by interfering with sex steroid hormone levels. Bioaccumulation of heavy metals affects different body districts directly, by altering cell viability and function, and indirectly, such as by increasing levels of oxidative stress. Thus, the presence of high amounts of toxic metals leads to an increase in ROS, but also to the depletion of intracellular antioxidant resources and the impairment of the functionality of some detoxifying enzymes 29 . In this context, heavy metal exposure has been shown to significantly inhibit mRNA expression levels of MT1E and CAT , which represent enzymes that can defend the cell against oxidative stress 3 . Based on these data, our study aimed to investigate how levels of certain heavy metals in the circulation of OPsis related to altered expression of 3 detoxifying genes, NQO1, CAT , and MT1E. Our analysis shows that in OPs, the levels of metals observed in the bloodstream correlates with a deregulation of MT1E and CAT gene expression. These findings are in agreement with reports in the literature by Azizieh and colleagues, who found decreased serum levels of 5 markers of oxidative stress, including CAT, correlated with decreased BMD levels in OPs group 30 . In addition, Pb, Hg, and Cd levels have been reported to correlate with decreased MT1 expression levels in heavy metal-exposed group, in agreement with our data 3 . Finally, further analysis identified a statistically significant direct correlation between Cu levels and CAT and MTE1 expression levels in CTRs and between Cu levels and MTE1 expression in OPs. These data show that Cu may play a predominant role in modulating the expression of both detoxifying enzymes, suggesting how this metal plays an important role in maintaining homeostasis. Overall, the results of this study broaden the landscape of knowledge on the role of heavy metals in the pathophysiology of osteoporosis. The present study presents a possible bias in the selection of individuals, as healthy subjects present a lower average age than OPs. However, this bias is not easily resolved due to the difficulty of recruiting older individuals without age-related bone diseases and other comorbidities. Further studies to be conducted on a larger cohort of OPs will be needed to validate these preliminary data. In addition, the analysis of other biological matrices, including blood and bone tissue, could better clarify the effect of metal exposure on the onset of musculoskeletal disease. Investigating the mechanisms of impact of contaminant on human health is, however, crucial, especially in association with multifactorial diseases such as osteoporosis.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.